The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear.

Abstract

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.

(A) Schematic diagram showing the strategy for generating Jag1flox mice. The targeting vector was designed to insert loxP sites (black arrowheads) on either side of exon 4, the DSL domain-encoding exon (white area in exon 4). The neomycin resistance cassette (for positive selection) was flanked by FRT sites (gray arrowheads) so that it could later be removed by crossing to FLPe-expressing mice. A diphtheria toxin gene was included for negative selection. Dotted lines depict the recombination events that occur when either the FLPe or Cre recombinases are present. Primer positions for genotyping are shown as small black arrows (a, DSLF; b, J1LoxR1; c, J1FlpF1; see for sequences). DT, diphtheria toxin; R, EcoR1; X, Xba1.(B) Southern blot analysis of EcoRI-digested DNA from ES cells using the external probe shown in (A). Left lane shows the wild-type band (12.3 kb), and the center and right lanes show correctly targeted ES cells that have both a wild-type band and a smaller mutant band (9.4 kb).

(A–D) Low- and high-power views of E10 embryos processed for whole-mount in situ hybridization using a Jag1 exon 4-specific probe. White arrows (A) point to the Jag1 signal in the otocyst (left arrow) and the eye (right arrow), two structures where Cre recombinase is expressed. In Jag1-cko mutants at E10, this signal is either absent or extremely weak. Black arrows (A and B) point to expression in the spinal cord and nephric duct, regions where Cre recombinase expression has not been reported in Foxg1-Cre mice. However, expression is consistently weaker in these areas in Jag1-cko embryos, indicating that there may be low levels of widespread expression of Cre recombinase in Jag1-cko embryos. In (D), the otocyst and the eye are outlined by a dotted line. Very little expression is observed in these regions, consistent with Foxg1-Cre expression.(E and F) In situ hybridization of E16.5 cochleae demonstrating Jag1 expression in wild-type (E) and Jag1-cko cochleae (F), where expression is entirely absent. Scale bars = 500 μm.

Scanning electron micrographs demonstrating the different patterns of hair cell production along the length of the cochlea in Jag1-cko embryos.(A–D) Low-power views of the apical and basal cochlear turns. The boxed-in area along the base in (A) and (B) is shown at higher magnification in (C) and (D). Note the absence of hair cells in the base of the Jag1-cko cochlea, except for a small patch of cells in the more apical portion (arrow). Scale bars = 500 μm.(E and F) In the midbasal region, more hair cells are observed, but they are arranged in patches, with no clear distinction between inner and outer hair cells.(G and H) In the apical turn, hair cells are continuous but generally arranged in only two rows. Scale bar = 100 μm.

Immunocytochemistry using two markers, myosin VIIA (red; all hair cells) and S100a (green; inner hair cells, Dieter's cells, and inner phalangeal cells) demonstrate patterns of hair and supporting cell production at E18.5 in control and Jag1-cko inner ears.(A–F) Sections through the indicated turns of the cochlea. Note the different hair cell patterns in the apical, middle and basal turns of the Jag1-cko cochlea. Normal morphology is shown (A) along with labeled structures, as follows: GER, greater epithelial ridge; IHCs, inner hair cells (color-coded yellow); LER, lesser epithelial ridge; OHCs, outer hair cells (color-coded red); SCs, supporting cells (color-coded green).(G–J) Patterns of hair and supporting cell production in the vestibular system. The utricular macula is extremely small with very few hair cells (H) while the saccule (J) shows robust hair and supporting cell production although the shape of the organ is smaller and malformed.

Early Analysis of the Patterns of Differentiation in the Jag1-cko Cochlea Indicates the Defects Are Caused by a Failure in the Formation of Sensory Cells and Not Subsequent Degeneration

Lectin staining of whole-mount cochlea at E16.5.(A) Normal patterning in wild-type control cochlea. GER, greater epithelial ridge.(B) Both the basal and middle portions of the cochlea are shown, although because it is much longer in the control (A), the very basal portion of the cochlea has been removed. Note the lack of hair cells in the basal portion of the Jag1-cko cochlea.(C–H) Boxed-in areas of (A) and (B) are shown at higher magnification in (C) and (D). Arrows in (D) indicate the abnormal patches of hair cells also observed at E18.5. Similarly, the boxed-in regions of (E) and (F) are shown at higher magnification in (G) and (H), demonstrating the few hair cells that are just beginning to differentiate in this region in both the control and the mutant (arrowheads). Scale bars = 500 μm for the corresponding panels.

At E14.5 JAG1 Is Expressed Directly adjacent to the Prosensory Domain That Is Disrupted in Jag1-cko Inner Ears

Immunocytochemistry at E14.5 using two markers of the prosensory domain, Sox2 and p27Kip1, in combination with JAG1 in both the basal and apical turns of the cochlea. Note that the JAG1 domain (red) does not overlap the p27Kip1 domain (green) (A and D), whereas the SOX2 domain does largely overlap with p27Kip1 (yellow) (B and E). Both SOX2 and p27Kip1 are down-regulated in the Jag1-cko cochlea (C and F), although there is weak expression of both markers in the apex (F). GER, greater epithelial ridge; LER, lesser epithelial ridge.

At E12.5 JAG1 Is Expressed within the Prosensory Domain and SOX2 Expression Is Down-Regulated within This Domain in Jag1-cko Cochleae

(A, B, D, and E) Alternate sections from a control embryo processed for immunocytochemistry using antibodies against either JAG1 or SOX2. Note the similar domain occupied by both JAG1 and SOX2 in the base of the cochlea (A and B; brackets). In the apical region, the two proteins are not colocalized (D and E). SOX2 is not expressed in the basal portions of the Jag1-cko cochlea (C) and shows only weak expression in the apex (F). bv, blood vessel; cd, cochlear duct.